Mapping densely packed αIIbβ3 receptors in murine blood platelets with expansion microscopy

Abstract

Interrogating platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting, a process that can save lives when stopping blood loss after an injury, but also kill when causing heart attack, stroke, or pulmonary embolism. The underlying key receptor distributions and interactions, in particular the relevance of integrin clustering, are not fully understood is because of highly abundant and densely distributed αIIbβ3 receptors. This makes receptor distributions difficult to assess even by super-resolution fluorescence microscopy. Here, we combine dual-color expansion and confocal microscopy with colocalization analysis to assess platelet receptor organization without the need of a super-resolution microscope. We show that 4x expansion is highly straight-forward for super-resolution microscopy of platelets, while 10x expansion provides higher precision at the price of increased efforts in sample preparation and imaging. Quantifying various receptor colocalization scenarios we demonstrate that expansion microscopy can pinpoint receptor distributions and interactions in resting and activated platelets being superior to conventional methods that fail in such dense 3D scenarios with highly abundant receptors. We reveal the presence of αIIbβ3 clusters in resting platelets, as well as in activated platelets, indicating that they contribute to the rapid platelet response during platelet clotting.

Keywords:

• Blood platelets
• colocalization
• integrin
• receptor mapping
• super-resolution microscopy

Acknowledgements

K.G.H., D.S., B.N., and H.S.H conceived and designed the experiments. H.S.H. performed the simulations for the resting platelets and supervised the ExM experiments. M.A. developed the platelet expansion protocol and the image acquisition procedure and developed the image processing and colocalization analysis procedures. M.A. and S.M. performed the ExM experiments and analysis on resting platelets. P.G. performed the ExM experiments on activated platelets while L.M.C.E. performed the data analysis. C.K. and M.M. performed the dSTORM imaging and cluster analysis. P.G. and S.M. performed the confocal imaging on activated and resting platelets respectively. V.G. screened the antibody incubation times for live and post-fix labelling and assisted with the animal handling. D.S., B.N., and V.G. advised with the platelet preparation and immunolabeling. B.N. provided the platelet antibodies. K.G.H., H.S.H and D.S. wrote the paper. All authors read and approved the final manuscript. We thank Shazeb Ahmad and Christoph Kempe for assistance in protocol development of ExM, Dr. Katharina Remer for support with documentation and management of animal experimentation and welfare, as well as Harald Schulze and Markus Sauer for fruitful discussion around platelet biology, expansion microscopy and colocalization analysis. Thanks to A. Balakrishnan for proof-reading.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

This study was supported by the Deutsche Forschungsgemeinschaft (SFB/TR 240, project number 374031971, B06 to D.S.; individual project HE 6438/4-1 to K.G.H. and NI 556/13-1 to B.N.), the Medical Faculty and the Graduate School of Life Sciences GSLS of the University of Würzburg (doctoral stipend to M.A. and S.M.), and the Rudolf-Virchow-Center of the University of Würzburg (H.S.H.).